A structural foundation for studying chlamydial polymorphic membrane proteins

ABSTRACT Polymorphic membrane proteins (Pmps) are chlamydial cell surface proteins with signature repeating FxxN and GGA (I, V, L) tetrapeptide motifs. These autotransporter proteins are key immune targets and mediators of host-microbe interactions but by unknown mechanisms. AlphaFold predictions reveal that Pmp passenger domains have two distinct β-helical parts: a C-terminal globular “Pmp middle region” that forms six β-helical rungs, and an N-terminal highly-regular “Pmp repeat region” that is from 9 to 25 rungs long. In the repeat region, paired FxxN and GGA (I, V, L) motifs – or close variants – are fully buried and packed together to form novel β-helical rungs from which variable-length loops extend out from one face. Similar motifs in proteins from diverse organisms are predicted to form analogous structures. In Pmps, the structures imply that the tetrapeptide motifs are not directly involved in host-cell adhesion, but position protein loops on a common face where they can interact with ligands important for infection. These structural descriptions provide a guide for mutagenesis studies to identify those parts of Pmps involved in infection. IMPORTANCE Infections by bacteria in the genus Chlamydia cause a range of widespread and potentially debilitating conditions in humans and other animals. We analyzed predicted structures of a family of proteins that are potential vaccine targets found in all Chlamydia spp. Our findings deepen the understanding of protein structure, provide a descriptive framework for discussion of the protein structure, and outline regions of the proteins that may be key targets in host-microbe interactions and anti-chlamydial immunity.


Supplemental Figures, Debrine et al.
Table S1.Pairwise percent amino acid sequence identities for the nine C. trachomatis D/UW/3 Pmp proteins.The upper right half of the matrix shows pairwise amino acid identities for each intact Pmp.The lower left half shows pairwise identities for the passenger domain only.The alignments on which these identities were based were conducted using Clustal Omega (1; https://www.ebi.ac.uk/Tools/msa/clustalo/), with no subsequent manual editing.The Uniprot IDs for the nine proteins are: PmpA -O84417, PmpB -O84418, PmpC -O84419, PmpD -O84818, PmpE -O84877, PmpF -P38008, PmpG -O84879, PmpH -O84880, PmpI -O84882.A full neighbor-joining rooted phylogenetic tree is shown without distance corrections.This tree is built with the same Pmps shown in Fig. 2. The tree is rooted with an autotransporter protein from Heliobacter pylori (accession number: P55981.1), a member of autotransporter phylogenetic group 8 (2).The scale bar represents the estimated number of substitutions per site.Boxed in purple are the CtPmps.An equivalent comparison of the same proteins but with the autotransporter domain removed yielded a similar tree, with all proteins having at least one CtPmp with which they shared a clade.

Figure S3. Comparison of the PmpE repeat region topology (A) with the amino acid sequence (B). A.
Topology model as shown in Fig 4A .B. The amino acid sequence of the PmpE passenger domain organized to show amino acid placement in the topology diagram.In both images, the N-terminus is near the bottom, and the C-terminus of the passenger domain is near the top.Highlighted are the division between the middle and repeat-containing region (dotted line), line rung numbers, residue numbers that initiate each rung and end the domain, β-strands (colors as in panel A), helices (underlined), and lengths of W-loops in place of amino acid sequence.Bold violet numbering is used for the W-loops corresponding to VR1 -VR5 regions that correlate with tissue tropism (3).The letters a -k listed below Rung 9 identify the positions with side chains pointing inwards in each rung of the regular repeat portion of the domain (see Figure 5A).

Figure S4. Detailed presentation of the repeat region rung structure in the predicted structure of all of
CtPmps A. Rungs 1 through 4 of PmpE with β-strand coloring as in Fig. 5A.The T5 turn is positioned at the front.In contrast to Figures 1B and 4B, the β-helix is rotated 180˚ around the helix axis, positioning the T3 turns and W-loops on the left and the β2-T2 segment, containing the FxxN motif, is positioned at the back right to left.Noteworthy in this image is that the β-helix does not rise like a corkscrew, but instead, has a step-like rise between rungs that largely occurs within the T5 turn.This image also demonstrates that within each rung, FxxN (starting at the end of β2) is at the same level as the following GGA(I,V,L) motif (ending at the start of β3).B. A stick model of 8 rungs of the repeat region β-helix axis of CtPmpE (residues 80-340, from Rung 9 to Rung 2) viewed from the amino-terminal end of the structure.This image shows the consistent positioning of the side chains of the nine inward facing residue positions (labeled a,b,c,d,e,f,i,j,k) and consistency in overall rung structure.The FxxN (red) and GGA(I,V,L) motifs are highlighted.C. Rung 0 through Rung 3 of PmpE using the same view as in Panel A, but with the front section cut away to reveal the β2-T2 FxxN side chains in the helix core (labeled in Rung 2).Highlighted are hydrogen bonds (H-bonds) that build the β2 and β3 sheets (yellow dashed lines) and those involving the Asn of the FxxN motif and the conserved Asp side chain from Rung 0 (cyan dashed lines).The Asn side chains form an internal "asparagine ladder", with each Asn making four H-bonds, two that link it with the Asn residues on the previous and following rungs, and two that link it to backbone carbonyl and NH groups in the next Rung.At the top of the panel is the Asp side chain from Rung 0 conserved in all chlamydial Pmp sequences which makes H-bonds with Rung 1 residues to seed the overall b-helix formation.Also labeled at the top left edge of the figure is the β2b strand (green arrow with "β2a seed" label) from Rung 1 that makes a unique antiparallel sheet interaction with the β2a strand from Rung 1 in a way that appears to seed that sheet as the protein folds from the C-toward the N-terminus.These featured interactions are present in each of the predicted CtPmp structures.

Figure S5. Models of chlamydial Pmp repeat regions with accompanying tables of motif and Ω-loops.
Alphafold2 predicted models of the CtPmps.A. Ribbon diagram for the predicted passenger domain structure of CtPmpA from D/UW/3, adjacent to a summary table of the motifs and Ω-loops present in each the rung of its regular repeat portion.The information is as described in Figure 6, highlighting both canonical FxxN (red) and GGA(I,V,L) (blue) motifs and non-canonical FxxN (magenta) and GGA(I,V,L) (cyan) motifs.B-H.Each panel has equivalent information to panel A, but is focused on PmpB, C, D, F, G, H, and I, respectively.I.A similar representation for C. pneumoniae Pmp20.

Figure S6
. Experimentally-determined structures most similar to segments of the CtPmpE passenger domain.A. Rungs 5-7 (residues 158-246) of CtPmpE (green Ca trace) were overlayed with the top hit (orange Ca trace, thick for aligned residues and thin for unaligned residues) from a DALI search (4) of CtPmpE Rungs 5-7 against the "PDB90" representative set of Protein Data Bank (PDB; 5) structures.The top hit was PDB entry 4peu chain A described as a "lyase-like protein from Clostridum thermocellum" and the comparison statistics were: Z-score = 4.5, percent sequence identity = 15%, and 75 aligned residues out of 250.The view of the rungs is as in Figure 5A, and the b-strands b1, b2, and b3 of the CtPmpE rungs are identified.B. The same as panel A but for a search using the entire CtPmpE repeat region (residues 18-407).The top hit for this search was PDB entry 5ny0 chain A described as "SRRP binding Region from Lactobacillus Reuteris", and the comparison statistics were: Z-score =19.7, percent sequence identity = 12%, and 262 aligned residues out of 343.C.The same as panel A but for a search using the whole CtPmpE middle region (residues 407-659).The top hit for this search was PDB entry 3sze chain A described as a "serine protease from E. coli", and the comparison statistics were: Z-score = 8.7, percent sequence identity = 12%, and 133 aligned residues out of 943.In addition to a view down the b-helix (left-hand image) as in panel A, an orthogonal view is shown (right hand image; similar to the view in Figure 1B) to clearly show how the best hit structure does possess the wider, more globular region that encompasses Rungs -4, -5, and the b-hairpin at the C-terminal end of the Pmp middle region.

Figure S2 .
Figure S2.A full phylogenetic tree of Pmp proteins from 10 Chlamydia spp.A full neighbor-joining rooted phylogenetic tree is shown without distance corrections.This tree is built with the same Pmps shown in Fig.2.The tree is rooted with an autotransporter protein from Heliobacter pylori (accession number: P55981.1), a member of autotransporter phylogenetic group 8 (2).The scale bar represents the estimated number of substitutions per site.Boxed in purple are the CtPmps.An equivalent comparison of the same proteins but with the autotransporter domain removed yielded a similar tree, with all proteins having at least one CtPmp with which they shared a clade.

Figure S7 .
Figure S7.Examples of proteins outside of chlamydia that contain paired FxxN and GGA(I,V,L) motifs.A. Alphafold2 predicted model of residues 0-430 I of a Phycisphaerae Myelin-associated glycoprotein (MAG): hypothetical protein (accession number: MBL7186538.1) and to the right a view down three rungs of its regular β-helix (residue numbers given in the figure) with canonical FxxN (red) and GGA(I,V,L) (blue) and noncanonical GGA(I,V,L) motifs (cyan) highlighted.B-E.The same as panel A but for the following: residues 0-280 of the Trichomonas vaginalis putative polymorphic outer membrane protein (POMP) (accession number: EAY21870.1) in panel B; residues 0-280 of the E. coli adhesin AidA-I_1 (accession number: A0A024L0S3) in panel C; residues 60-370 of the Fischerella ambigua POMP protein (accession: A0A076NA94) in panel D; and residues 74-213 of the Saprolegnia parasitica β-helix containing protein (accession number: A0A067CJ47) in panel E.

Fig S5 :Fig
Fig S5: Models of chlamydial Pmp repeat regions with accompanying tables of motif and Ω-loops.

Fig S7 :
Fig S7: Examples of proteins outside of chlamydia that contain paired FxxN and GGA(I,V,L) motifs.